Abstract
Purpose: :
Recent studies have shown that galectin-3 (Gal-3), a β-galactoside-binding lectin, promotes cell migration during re-epithelialization of corneal wounds. The goal of this study was to characterize the molecular mechanism by which Gal-3 stimulates cell migration.
Methods: :
The human corneal epithelial cells were exposed to recombinant human Gal-3 in serum-free media for 30 minutes and alteration in morphology of the cells was studied by fluorescence microscopy. After exposure to Gal-3 for various time periods, the activation of Rac1 and focal adhesion kinase (FAK) was examined by Western blot analyses. The Gal-3-binding proteins were isolated by affinity chromatography of cell lysates on a Gal-3-Sepharose column. Furthermore, the expression of β1,6N-acetylglucosaminytransferase V (Mgat5), an enzyme that synthesizes high-affinity glycan ligands for Gal-3, was knocked down by shRNA to evaluate the role of complex N-glycans on Gal-3-mediated lamellipodia formation.
Results: :
Exogenous Gal-3, but not Gal-1 or -8, promoted cell scattering and formation of lamellipodia in epithelial cells in a β-lactose-inhibitable manner. 3β1 integrin was identified as the major Gal-3-binding protein in corneal epithelial cells. Preincubation of cells with anti-3 integrin function-blocking antibody (P1B5) significantly inhibited the induction of lamellipodia by Gal-3. Furthermore, exogenous Gal-3 activated Rac1 GTPase, a signaling molecule well known for its role in the reorganization of actin cytoskeleton and formation of lamellipodial extensions. Exogenous Gal-3 also activated FAK. Experiments involving knockdown of Mgat5, revealed that carbohydrate-mediated interaction between Gal-3 and complex N-glycans on 3β1 integrin plays a key role in Gal-3-induced lamellipodia formation.
Conclusions: :
We propose that Gal-3 promotes epithelial cell migration during corneal wound closure by cross-linking Mgat5-modified complex N-glycans on 3β1 integrin and, subsequently activating 3β1 integrin-Rac1 signaling to promote lamellipodia formation.
Keywords: cornea: epithelium • wound healing